Coxiella burnetii is a Gram-negative, obligate intracellular bacterium that is the causative agent of Q-fever in humans and is further classified as a Class B select agent. After uptake by a host cell, C. burnetii subverts specific host cell processes and establishes a specialized intracellular compartment where the bacteria can survive and replicate. Modulation of the host cell during each step of infection is likely due to the action of bacterial effector proteins secreted from the C. burnetii Dot/Ion type IV secretion system. The long-term objectives of this application are to better understand the molecular interactions and processes that occur between C. burnetii and host cells during intracellular infection and to uncover novel properties that may result from a systemic view of C. burnetii pathogenesis. Both bacterial and host proteins are involved in the biogenesis of the C. burnetii containing vacuole;however, little is known about the mechanisms involved. The goal of Aim 1 is to use RNA interference and high-throughput fluorescence microscopy to identify then characterize human host factors important for intracellular replication of C. burnetii and establishment of the C. burnetii containing vacuole. Host factors identified by the screen will be tested for bacterial-dependent localization to the Coxiella-containing vacuole. The goal of Aim 2 is to use cell biological approaches to analyze individual stages of C. burnetii infection to determine where depletion of an important host protein disrupts the infection cycle. The goal of Aim 3 is to determine the role that C. burnetii secreted effector proteins play in interacting with host proteins important for intracellular growth. PUBLIC HEALTH RELEVANCE: Infection by intracellular bacterial pathogens leads to significant morbidity and mortality and represents a serious human health threat. Global systemic approaches are necessary to determine both the bacterial and host contributions to the mechanisms of disease at a molecular level. Analysis of Coxiella burnetii infection using genome-wide systems biology approaches can increase knowledge of these interactions and lead to innovative approaches to discover novel antimicrobial therapies for intracellular bacterial pathogens.